Routine Diagnostics Research Articles

Molecular Analysis of PIK3CA in Metastatic Hormone Receptor-Positive Breast Cancer in Chile: Clinical and Pathological Insights

Breast cancer is the most common cancer among women and a leading cause of cancer-related deaths. PIK3CA gene mutations, which are often present in advanced HR+ breast cancer, can be targeted by alpelisib. However, data on PIK3CA mutations in Chile are limited. Here, we aim to assess the mutational status of PIK3CA in metastatic breast cancer tissues from Chilean patients and describe their clinicopathological characteristics and survival outcomes. We analyzed 102 formalin-fixed, paraffin-embedded metastatic breast cancer samples from 96 patients diagnosed at three Chilean hospitals between 2007 and 2023. PIK3CA mutations were identified using targeted sequencing, and clinicopathological data were collected. We evaluated associations between mutational status, clinicopathological features, and survival. The median age at diagnosis was 56 years. The most common metastatic sites were liver (29.4%), bone (17.6%), and lung/pleura (16.7%). Most patients were HR+ HER2− (83.3%), with 57.3% showing HER2-low status. PIK3CA mutations were present in 40.6% of patients, mainly in exons 7, 9, and 20. No significant associations were found between PIK3CA mutations and clinicopathological characteristics or survival. Our study reveals a high frequency of PIK3CA mutations in HR+ metastatic breast cancer, consistent with global data. The majority of mutations are targetable with alpelisib. The proportion of HER2-low status patients suggests potential benefits from novel HER2-targeted therapies. These findings highlight the need for routine molecular diagnostics in Chile to improve personalized treatment and address economic and access challenges.

Infants < 90 days of age with late-onset sepsis display disturbances of the microbiome-immunity interplay.

We hypothesized that previously healthy infants < 90 days of age with late-onset sepsis (LOS) have disturbances of the gut microbiome with yet undefined specific immunological patterns. We performed a prospective single-center convenience sample study between January 2019 and July 2021 in a case-control design. Routine diagnostics included conventional cultures (blood, cerebrospinal fluid, urine), PCRs and inflammatory markers in infants aged < 90 days with clinical LOS. We additionally analyzed blood lymphocyte subsets including CD4 + CD25 + forkhead box protein (FoxP3)+ Tregs and performed 16S rRNA sequencing of stool samples, both compared to age-matched healthy controls. Results were adjusted for potential confounders that may influence microbial composition. 51 infants with fever and clinical LOS were enrolled. Bacterial sepsis was diagnosed in n = 24 (47.1%) and viral infection in n = 13 (25.5%) infants, whereas in 14 (27.3%) infants the cause of fever remained undetermined. When compared to healthy controls, the gut microbiome of LOS infants at disease onset was characterized by a shift in community composition, specifically, decreased abundance of B. longum and an increase of Bacteroidia spp. Intriguingly, the abundance of B. longum negatively correlated with the frequency of blood CD4-positive cells in healthy controls but not in infants with LOS. At one year of age, we observed microbiome differences in infants with history of LOS when compared to healthy controls, such as an increased gut microbial diversity. Our data suggest potential signatures of the microbiome-immunity interplay in infants with LOS, which should be investigated further as possible targets for prevention.

Prevalence of monoclonal proteins in patients with isolated hypogammaglobulinemia on serum protein electrophoresis

Isolated hypogammaglobulinemia (IH) is an electrophoretic pattern that can be encountered on serum protein electrophoresis (SPEP) and is defined as a decreased but morphologically normal γ-globulin fraction with normal α- and β-globulin fractions. SPEP is mainly used to detect monoclonal proteins which are usually observed as additional peaks in the electropherogram. However, they may also be more discretely present in a significant proportion of patients presenting with IH. Therefore, we aimed to evaluate i) via both retrospective and prospective analysis to what extent paraproteins as identified by immunofixation are present in patients demonstrating IH on SPEP and ii) whether other parameters may predict their presence in IH-patients. For this purpose, we first reviewed historic SPEP- and immunofixation results in our tertiary hospital and determined paraprotein prevalence in this retrospective cohort. This analysis showed immunofixation was requested in only 519/3938 (13.2%) historic IH-results with 52/519 (10%) patients demonstrating paraproteins. Next, various laboratory parameters were compared between paraprotein-positive and -negative patients and subjected to logistic regression models but regrettably, no parameter could be retained as promising predictor of paraproteins. Lastly, to confirm paraprotein prevalence seen in the historical query, we conducted a six-month prospective analysis during which immunofixation was requested more frequently in IH-cases during routine diagnostics and which showed paraproteins to be present in 20/83 (24.1%) of IH-patients. Hence, as up to 24% of patients with IH may harbour paraproteins, one should consider performing follow-up analyses (e.g. immunofixation, urine electrophoresis and/or free light chain analysis) for all IH-cases identified via SPEP.

Performance of LDL-C only compared to the Dutch Lipid Clinic Network Score for screening of familial hypercholesterolaemia: the Austrian experience and literature review.

Familial hypercholesterolaemia (FH) is a severely underdiagnosed, inherited disease, causing dyslipidaemia and premature atherosclerotic cardiovascular disease. In order to facilitate screening in a broad clinical spectrum, we aimed to analyse the current yield of routine genetic diagnostics for FH and to evaluate the performance of the Dutch Lipid Clinic Network Score (DLCNS) compared to a single value, the off-treatment LDL-cholesterol exceeding 190 mg/dL. We investigated all patients that underwent molecular genotyping routinely performed for FH over a 4-year period in two Austrian specialist lipid clinics. Variants reported in FH-causing genes including LDLR, APOB, PCSK9, LDLRAP, and APOE were collected and classified. For clinical classification, the DLCNS was calculated retrospectively and compared to the original scores documented in patient charts. Additionally, a literature review on comparisons of DLCNS to LDL-C was performed. Of 469 patients tested, 21.3% had a disease-causing variant. A median of 3 out of 8 (excluding genotyping results and LDL-C) DLCNS criteria were unavailable. DLCNS was documented in 48% of cases, with significant discrepancies compared to retrospective scoring (P < 0.001). DLCNS did not outperform off-treatment LDL-C alone (Δ = 0.006; P = 0.660), analogously to several reports identified in the literature. A single cut-off of 190 mg/dL LDL-C compared to DLCNS ≥ 6 showed excellent sensitivity (84.9% vs. 53.8%) and acceptable specificity (39.0% vs. 84.1%). Missing criteria and severe discrepancies observed between retrospective and on-site scoring by treating physicians were highly prevalent, confirming limited utility of DLCNS in clinical routine and warranting a single off-treatment LDL-C cut-off of 190 mg/dL for enhanced index-case identification.

Mechanistic Insights Into Redox Damage of the Podocyte in Hypertension.

Podocytes are specialized cells within the glomerular filtration barrier, which are crucial for maintaining glomerular structural integrity and convective ultrafiltration. Podocytes exhibit a unique arborized morphology with foot processes interfacing by slit diaphragms, ladder-like, multimolecular sieves, which provide size and charge selectivity for ultrafiltration and transmembrane signaling. Podocyte dysfunction, resulting from oxidative stress, dysregulated prosurvival signaling, or structural damage, can drive the development of proteinuria and glomerulosclerosis in hypertensive nephropathy. Functionally, podocyte injury leads to actin cytoskeleton rearrangement, foot process effacement, dysregulated slit diaphragm protein expression, and impaired ultrafiltration. Notably, the renin-angiotensin system plays a pivotal role in podocyte function, with beneficial AT2R (angiotensin receptor 2)-mediated nitric oxide (NO) signaling to counteract AT1R (angiotensin receptor 1)-driven calcium (Ca2+) influx and oxidative stress. Disruption of this balance contributes significantly to podocyte dysfunction and drives albuminuria, a marker of kidney damage and overall disease progression. Oxidative stress can also lead to sustained ion channel-mediated Ca2+ influx and precipitate cytoskeletal disorganization. The complex interplay between GPCR signaling, ion channel activation, and redox injury pathways underscores the need for additional research aimed at identifying targeted therapies to protect podocytes and preserve glomerular function. Earlier detection of albuminuria and podocyte injury through routine noninvasive diagnostics will also be critical in populations at the highest risk for the development of hypertensive kidney disease. In this review, we highlight the established mechanisms of oxidative stress-mediated podocyte damage in proteinuric kidney diseases, with an emphasis on a hypertensive renal injury. We will also consider emerging therapies that have the potential to selectively protect podocytes from redox-related injury.

Oxford Nanopore Technology-Based Identification of an Acanthamoeba castellanii Endosymbiosis in Microbial Keratitis

(1) Background: Microbial keratitis is a serious eye infection that carries a significant risk of vision loss. Acanthamoeba spp. are known to cause keratitis and their bacterial endosymbionts can increase virulence and/or treatment resistance and thus significantly worsen the course of the disease. (2) Methods and Results: In a suspected case of Acanthamoeba keratitis, in addition to Acanthamoeba spp., an endosymbiont of acanthamoebae belonging to the taxonomic order of Holosporales was detected by chance in a bacterial 16S rDNA-based pan-PCR and subsequently classified as Candidatus Paracaedibacter symbiosus through an analysis of an enlarged 16S rDNA region. We used Oxford Nanopore Technology to evaluate the usefulness of whole-genome sequencing (WGS) as a one-step diagnostics method. Here, Acanthamoeba castellanii and the endosymbiont Candidatus Paracaedibacter symbiosus could be directly detected at the species level. No other microbes were identified in the specimen. (3) Conclusions: We recommend the introduction of WGS as a diagnostic approach for keratitis to replace the need for multiple species-specific qPCRs in future routine diagnostics and to enable an all-encompassing characterisation of the polymicrobial community in one step.

Development and Validation of an Indirect and Blocking ELISA for the Serological Diagnosis of African Swine Fever

African swine fever (ASF) is an economically devastating viral disease of pigs caused by the ASF virus (ASFV). The rapid global spread of ASF has increased the demand for ASF diagnostics to be readily available and accessible. No commercial ASF enzyme-linked immunosorbent assay (ELISA) kits are manufactured and licensed in North America. Here, we report the development of two serological diagnostic assays, a blocking ELISA (bELISA) based on ASFV glycoprotein p54 and an indirect ELISA (iELISA) based on ASFV glycoproteins p54 and p72. The assays showed high sensitivity and specificity and detected anti-ASFV antibodies in serum samples from experimentally infected animals as early as 8 days post-infection. The two assays were produced commercially (AsurDx™ bELISA and iELISA) and subjected to extensive validation. Based on data from a set of characterized reference sera, the prototype commercial assays, while maintaining 100.00% specificity, showed 97.67% (AsurDx™ bELISA) and 83.72% (AsurDx™ iELISA) sensitivity. Both prototype assays detected anti-ASFV antibodies in serum samples collected from pigs experimentally infected with multiple ASFV strains and field samples collected from sick, recovering, and vaccinated animals. The two commercially available assays can be used in routine ASF diagnostics, serological surveys, and for evaluating serological responses to ASF vaccine candidates.

Role of Targeted Sequencing in Routine Diagnostics of Spitz Melanocytic Neoplasms-An Analysis of 70 Cases.

There is growing evidence that the Spitz group of melanocytic neoplasms should be restricted to those harboring kinase receptor fusions and HRAS mutations/11p15 amplification. The presence of genomic alterations characteristic of conventional melanomas (BRAF and NRAS mutations) precludes a diagnosis of a Spitz neoplasm. It is often challenging to distinguish Spitz neoplasms from conventional melanomas with spitzoid morphology on histopathological grounds alone. We report a series of 70 consecutive melanocytic tumors in which targeted sequencing was indicated to distinguish Spitz from spitzoid neoplasms and to classify Spitz neoplasms along the biological spectrum. Final diagnoses incorporating molecular results included 12 conventional melanomas (nine of which with NRAS mutations), five Spitz melanomas, 35 atypical Spitz tumors, eight Spitz nevi, three melanocytic tumors with a MAP2K1 mutation, and seven desmoplastic Spitz nevi/tumors. There were significant discrepancies between initial diagnoses and final diagnoses after incorporating molecular results in 24 (34%) cases, including nine conventional melanomas favored to be Spitz neoplasms and nine Spitz neoplasms favored to be conventional melanomas. It is often not possible to reliably distinguish Spitz neoplasms from spitzoid melanocytic tumors without identifying their driver genomic alterations. Applying next-generation sequencing in diagnostically problematic tumors improves diagnostic accuracy.

High-Resolution Melting PCR as a Fast and Simple Molecular Biology-Based Method for the Identification of Hypervirulent Clostridioides difficile Strains Directly in Stool Samples

Clostridioides difficile became one of the main causes of nosocomial infections in all clinical settings worldwide, especially among patients undergoing antibiotic therapy. The incidence and severity of C. difficile infections, from mild diarrhea to life-threatening pseudomembranous colitis, correlate with the spread of the hypervirulent binary toxin (CDT)-producing strains. The use of the real-time HRM-PCR method enables the identification of hypervirulent C. difficile strains directly in the diarrheal stool samples of patients suspected of being infected with this bacterium. For this purpose, the cdtA and cdtB genes encoding CDT subunits, as well as the species-specific gluD gene, were detected to identify the presence of this bacterium in the tested samples. The sensitivity, specificity, negative predictive value (NPV) and positive predictive value (PPV) of the established method were also assessed. The obtained results were compared with the results of eazyplex® C. difficile complete test (AmplexDiagnostics GmbH) based on the LAMP method, used in standard microbiological diagnostics. The values of the assessed diagnostic parameters for the detected genes ranged from 58.82% to 98.85%. The lowest value (58.82%) was obtained for the PPV of cdtB and the highest (98.85%) for the NPV of this gene. The real-time HRM-PCR method enables fast and simple detection of the investigated genes of hypervirulent C. difficile strains and, after careful optimization, may demonstrate high potential for usefulness in routine microbiological diagnostics.

Quantification of pulmonary edema using automated lung segmentation on computed tomography in mechanically ventilated patients with acute respiratory distress syndrome

BackgroundQuantification of pulmonary edema in patients with acute respiratory distress syndrome (ARDS) by chest computed tomography (CT) scan has not been validated in routine diagnostics due to its complexity and time-consuming nature. Therefore, the single-indicator transpulmonary thermodilution (TPTD) technique to measure extravascular lung water (EVLW) has been used in the clinical setting. Advances in artificial intelligence (AI) have now enabled CT images of inhomogeneous lungs to be segmented automatically by an intensive care physician with no prior radiology training within a relatively short time. Nevertheless, there is a paucity of data validating the quantification of pulmonary edema using automated lung segmentation on CT compared with TPTD.MethodsA retrospective study (January 2016 to December 2021) analyzed patients with ARDS, admitted to the intensive care unit of the Department of Anesthesiology and Critical Care Medicine, University Hospital Mannheim, who underwent a chest CT scan and hemodynamic monitoring using TPTD at the same time. Pulmonary edema was estimated using manually and automated lung segmentation on CT and then compared to the pulmonary edema calculated from EVLW determined using TPTD.Results145 comparative measurements of pulmonary edema with TPTD and CT were included in the study. Estimating pulmonary edema using either automated lung segmentation on CT or TPTD showed a low bias overall (− 104 ml) but wide levels of agreement (upper: 936 ml, lower: − 1144 ml). In 13% of the analyzed CT scans, the agreement between the segmentation of the AI algorithm and a dedicated investigator was poor. Manual segmentation and automated segmentation adjusted for contrast agent did not improve the agreement levels.ConclusionsAutomated lung segmentation on CT can be considered an unbiased but imprecise measurement of pulmonary edema in mechanically ventilated patients with ARDS.

Analysis of a persistent outbreak with vancomycin resistant Enterococcus faecium (VREfm) revealed the need for an adapted diagnostic algorithm

Our setting was challenged with an outbreak of different vancomycin resistant Enterococcus faecium (VREfm) including vanA and/or vanB containing isolates. Remarkably, it was observed that screening by use of a vanA and vanB real-time PCR on overnight enriched specimens from time to time tested positive for VanB with very low Ct-values, whereas VREfm-specific enrichment cultures remained negative. Here, we describe the analysis of the diagnostic results leading to adaptation of the diagnostic algorithm. Per specimen of each patient, results of the vanA and vanB screening PCR and of the VREfm-specific culture (Brilliance VRE) were collected and combined with genotyping data of the identified VREfm isolates. During the outbreak, a second VREfm-specific culture medium (CHROMagar VRE) was introduced, and results were compared to the results obtained with Brilliance VRE agar. Thirty-five patients were identified as VREfm-carrier, in which four different strains were identified comprising vanA (STnew-CT7088) and/or vanB (ST80-CT1065, ST117-CT7117 and ST117-CT7118). Complementing results of PCR, culture and genotyping revealed that culture with Brilliance VRE agar was inadequate for detection of the vanB ST117 isolates identified, irrespective of vancomycin MIC values. In contrast, CHROMagar VRE was able to correctly detect these vanB ST117 isolates and other tested isolates. Our data showed that the vanB ST117 containing isolates were inadequately detected by the VREfm-specific culture media, possibly contributing to unnoticed spread of VREfm. For this reason, CHROMagar VRE was evaluated during the outbreak and subsequently implemented in routine diagnostics, replacing Brilliance VRE agar.

Breast cancer survival prediction using an automated mitosis detection pipeline.

Mitotic count (MC) is the most common measure to assess tumor proliferation in breast cancer patients and is highly predictive of patient outcomes. It is, however, subject to inter- and intraobserver variation and reproducibility challenges that may hamper its clinical utility. In past studies, artificial intelligence (AI)-supported MC has been shown to correlate well with traditional MC on glass slides. Considering the potential of AI to improve reproducibility of MC between pathologists, we undertook the next validation step by evaluating the prognostic value of a fully automatic method to detect and count mitoses on whole slide images using a deep learning model. The model was developed in the context of the Mitosis Domain Generalization Challenge 2021 (MIDOG21) grand challenge and was expanded by a novel automatic area selector method to find the optimal mitotic hotspot and calculate the MC per 2 mm2. We employed this method on a breast cancer cohort with long-term follow-up from the University Medical Centre Utrecht (N = 912) and compared predictive values for overall survival of AI-based MC and light-microscopic MC, previously assessed during routine diagnostics. The MIDOG21 model was prognostically comparable to the original MC from the pathology report in uni- and multivariate survival analysis. In conclusion, a fully automated MC AI algorithm was validated in a large cohort of breast cancer with regard to retained prognostic value compared with traditional light-microscopic MC.

Validation of Keratin 17 as a Tissue Biomarker in the Diagnosis of Upper Tract Urothelial Carcinoma.

Upper tract urothelial carcinoma (UTUC) has a relatively low incidence but presents significant surveillance and treatment challenges. Therefore, novel biomarkers for the accurate detection of upper tract urothelial tumors are urgently needed. We evaluated the expression of Keratin 17 (KRT17), an oncoprotein implicated in the cell cycle progression of multiple human cancers and previously studied in bladder urothelial carcinoma, by immunohistochemistry in 139 UTUC cases, including noninvasive, invasive papillary urothelial carcinoma and urothelial carcinoma in situ. KRT17 expression pattern (basal/negative vs. nonbasal) and H-score were evaluated. The expression pattern was significantly different in normal (NL) compared to malignant urothelium. Nonbasal KRT17 expression was significantly higher in pTa (p<0.001) and invasive (pTinv) (p=0.0023) urothelial carcinoma compared to NL, and in pTinv compared to pTa (p=0.0391). Sensitivity and specificity for distinguishing benign from malignant tumors were 85% and 82, respectively, with an area under the curve of 0.83 (p <0.001). The KRT17 H-score was significantly higher in pTa and pTinv compared to NL (p < 0.001 and p=0.0035, respectively). Sensitivity and specificity for distinguishing benign from malignant carcinoma were 91% and 69%, respectively, with an AUC of 0.81 (p=0.0010). KRT17 was not associated with tumor site, grade, or stage. In summary, K17 is a sensitive and specific marker of neoplastic upper tract urothelium, and its potential use in routine diagnostics should be explored in larger studies.

Ramming the parasites: Evaluation of quarantine procedures against Haemonchus contortus at sheep markets in Sweden

In Sweden, it is recommended to treat rams at sheep markets with ivermectin and then keep them in quarantine to minimise the risk of transmission of Haemonchus contortus between farms. The aim of this study was to evaluate the effectiveness of this practise. Accordingly the gastrointestinal parasite status of 50 rams purchased at two sheep markets in central Sweden in autumn 2019 was investigated using faecal samples collected on the day of sale (test 1), 12 ± 3 days after ivermectin injection (test 2, in quarantine) and a few months later before inclusion in the new flocks (test 3). We used both traditional diagnostic methods (i.e. identification of nematode eggs in faeces or larvae in cultures when H. contortus eggs could not be identified) and a molecular test based on the digital droplet PCR platform to further identify positive samples. In test 1, conducted in autumn, 40 % of rams were FEC-positive, but only 12 % (six rams) were infected with H. contortus according to conventional routine diagnostics. In test 2, 8 % (four rams) were FEC-positive, including one with 1050 eggs, 90 % of which were identified as H. contortus. This ram was therefore returned to the supplier. However, ivermectin was found to be effective when this animal and a group of lambs from the same farm were treated and tested again. Test 3 was performed on 44 of the same rams (in addition to the returned ram, two rams died in quarantine and the samples from three rams were never provided by the owners). The proportion of FEC-positive animals was 42 %, with an even higher proportion (27 %) of animals being H. contortus-positive than in test 1. The corresponding results for tests 1, 2 and 3 with the ddPCR assay were 18 %, 4 % and 76 %, respectively. This study demonstrates the superiority of DNA detection over microscopy, which is the mainstay in most diagnostic laboratories. Although the combined results confirm that H. contortus survived quarantine in two rams, in the other cases it is not clear whether the spring infection rates are due to re-emergence of persistent larvae from quarantine or reinfection on the new farm. These results suggest not only that we should recommend that sheep farmers use a more sensitive molecular test when purchasing and introducing new animals to their flock, but also that the reliability of injectable ivermectin as a quarantine treatment for removal of adult and larval stages needs further investigation.

Detecting Dirofilaria immitis: Current Practices and Novel Diagnostic Methods

The nematode Dirofilaria immitis is responsible for a vector-borne disease affecting canines and humans worldwide, known as cardiopulmonary dirofilariasis. An accurate and early diagnosis is of the utmost importance for effective disease management. While traditional microscopy-based methods remain invaluable, they have inherent limitations. Serological tests, in particular ELISA and immunochromatographic tests, are employed due to their capacity to detect D. immitis antigens, offering ease of use and diagnostic accuracy. The advent of molecular methods has the potential to enhance routine diagnostic approaches, with polymerase chain reaction (PCR) and real-time PCR (qPCR) becoming the most prevalent techniques. Despite not yet being integrated into routine diagnostics, which are predominantly based on the Knott’s test and serological methods, these techniques offer significant benefits in the context of scientific research. This article proceeds to examine the potential of advanced techniques, such as high-resolution melting qPCR (HRM-qPCR), loop-mediated isothermal amplification (LAMP), droplet digital PCR (ddPCR), and microRNA (miRNA) detection, which are capable of enhanced sensitivity and early detection. The following work provides an in-depth analysis of the various diagnostic methods, emphasising the necessity of the continuous improvement and adaptation of these tools to effectively combat D. immitis. The findings underscore the importance of integrating these advanced methods into routine practice to improve detection rates and outcomes for infected animals.

An analysis of acid-labile subunit (ALS) levels in children with short stature born with normal weight.

The acid-labile subunit (ALS) is a protein best known for its function in stabilising the insulin like growth factor-1/2-insulin-like growth factor-1 binding protein 3/5 (IGF-1/2-IGFBP3/5) binary complex by creating the ternary complex and in consequence regulating the biological activity of IGF-1. The aim of the study was to assess ALS concentrations in a chosen population of children with short stature taking into account their clinical diagnosis. A total of 109 prepubertal children were involved in the study - 85 children in the study group and 24 in controls. In all the children IGF-1, IGFBP3, and ALS were measured. The study group was divided according to diagnosis into groups: growth hormone deficiency (GHD), constitutional delay of growth and puberty (CDGP), idiopathic short stature (ISS), and familial short stature (FSS). In the control group the ALS concentration ranged from 4.81 to 13.66 μg/mL. In the whole study group the ALS concentration ranged from 2.73 to 15.81 μg/mL. The difference between both groups was statistically significant (p < 0.0001, R = 0.39). A strong, statistically significant correlation between ALS levels and age was observed, but only in the study group (p < 0.0001, r = 0.59). The ALS standard deviation score (SDS) was not significantly different between the control and CDGP children (p = 0.0644). The ALS concentration was significantly lower in children with short stature. There was, however, no difference between the subgroups of the study group. There was no significant difference in ALS SDS between the control group and children with constitutional delay of growth and development. The usefulness of ALS in routine short stature diagnostics is uncertain, but it might play a role in the diagnosis of children with ISS and CDGP in the future.

Cerebrospinal fluid red blood cells and total protein are associated with clinical outcome in spontaneous subarachnoid hemorrhage.

Prognostication in patients with spontaneous subarachnoid hemorrhage (SAH) can be challenging. The aim of this study was to assess whether cerebrospinal fluid (CSF) red blood cell (RBC) count and total protein (TP) concentration are associated with SAH prognosis. Patients with SAH treated at the neurological intensive care unit (ICU) in Innsbruck were included in this real-world, observational study. Longitudinal CSF samples were collected as part of routine diagnostics. RBC count and CSF TP at the time of admission (RBCfirst, TPfirst), in Week 1 (RBCDays1-7, TPDays1-7), Week 2 (RBCDays8-14, TPDays8-14), and Week 3 or thereafter (RBCDay>14, TPDay>14), the highest detected value (RBChighest, TPhighest), as well as the RBC count adjusted for disease duration (RBCadjusted) were assessed. Primary outcomes were good functional outcome after 3 months, defined as modified Rankin scale score ≤2 and ICU survival. A total of 183 SAH patients with a female predominance (69%), a median (interquartile range [IQR]) age of 60 (50-70) years and median (IQR) Hunt and Hess score of 4 (3-5) were included. Multivariable analyses revealed that lower values of RBCfirst, RBCadjusted, RBChighest, TPfirst and TPhighest were associated with good functional outcome and hospital survival. Lower TP concentrations in Weeks 1, 2 and 3 were associated with good functional outcome, and in Weeks 1 and 2 with ICU survival. Early RBC measurements (Week 1) were associated with good functional outcome and ICU survival. Low CSF RBC counts and TP concentrations were associated with good functional outcome and ICU survival in a real-world cohort of SAH patients requiring external ventricular drainage.

Reduced vancomycin susceptibility in Staphylococcus aureus clinical isolates: a spectrum of less investigated uncertainties

BackgroundStaphylococcus aureus clinical isolates with vancomycin MICs of 2 µg/ml have been associated with vancomycin therapeutic failure and the heterogenous vancomycin-intermediate S. aureus (hVISA) phenotype. While carriage of van genes has usually been associated with higher level of MIC and frank vancomycin resistance, the unrecognized risk of hetero-resistance is frequently underestimated. Methods used for assessing vancomycin susceptibility have also shown different concordance and variable performance and accessibility in routine clinical diagnostics posing a challenge to inform treatment selection in hospital settings.MethodsA total of 195 clinical samples were obtained among which 100 S. aureus isolates were identified. Ninety-six MRSA isolates have been identified using cefoxitin disc and mecA gene detection. The vanA and vanB genes have been screened for in the studied isolates using conventional PCR amplification. Examination of reduced vancomycin susceptibility has been performed using vancomycin screen agar, Broth Micro Dilution method (BMD), and VITEK2. Blood isolates were screened for hVISA using PAP-AUC method.ResultsVancomycin screening agar applied to 96 MRSA isolates revealed 16 isolates with reduced vancomycin susceptibility. Further MIC testing revealed that 7 isolates were VISA and only 1 isolate was identified as VRSA using both BMD MIC method and VITEK2. Among 24 tested blood isolates, 4 isolates (16.7%) revealed the hVISA phenotype as identified using PAP-AUC method. Using PCR, vanA gene was identified in 5 S. aureus isolates (5%). Three of them were VSSA while the other two isolates were VISA.ConclusionIn this study, we report the very low prevalence of VRSA among the tested S. aureus clinical isolates (1%) and the existence of hVISA phenotype among studied S. aureus blood isolates at the rate of 16.7% in our setting. Fifty percent (8/16) of isolates that demonstrated reduced vancomycin susceptibility using vancomycin agar screen tested susceptible using both broth dilution method and VITEK2. These finding together with the concerning silent carriage of vanA gene among VSSA and VISA (5%) may underly hidden and uninvestigated factors contributing to vancomycin treatment failure that warrant cautious vancomycin prescription.

Identification of novel 3D-genome altering and complex structural variants underlying retinitis pigmentosa type 17 through a multistep and high-throughput approach.

Autosomal dominant retinitis pigmentosa type 17 (adRP, type RP17) is caused by complex structural variants (SVs) affecting a locus on chromosome 17 (chr17q22). The SVs disrupt the 3D regulatory landscape by altering the topologically associating domain (TAD) structure of the locus, creating novel TAD structures (neo-TADs) and ectopic enhancer-gene contacts. Currently, screening for RP17-associated SVs is not included in routine diagnostics given the complexity of the variants and a lack of cost-effective detection methods. The aim of this study was to accurately detect novel RP17-SVs by establishing a systematic and efficient workflow. Genetically unexplained probands diagnosed with adRP (n = 509) from an international cohort were screened using a smMIPs or genomic qPCR-based approach tailored for the RP17 locus. Suspected copy number changes were validated using high-density SNP-array genotyping, and SV breakpoint characterization was performed by mutation-specific breakpoint PCR, genome sequencing and, if required, optical genome mapping. In silico modeling of novel SVs was performed to predict the formation of neo-TADs and whether ectopic contacts between the retinal enhancers and the GDPD1-promoter could be formed. Using this workflow, potential RP17-SVs were detected in eight probands of which seven were confirmed. Two novel SVs were identified that are predicted to cause TAD rearrangement and retinal enhancer-GDPD1 contact, one from Germany (DE-SV9) and three with the same SV from the United States (US-SV10). Previously reported RP17-SVs were also identified in three Australian probands, one with UK-SV2 and two with SA-SV3. In summary, we describe a validated multi-step pipeline for reliable and efficient RP17-SV discovery and expand the range of disease-associated SVs. Based on these data, RP17-SVs can be considered a frequent cause of adRP which warrants the inclusion of RP17-screening as a standard diagnostic test for this disease.

Undergraduate musculoskeletal ultrasound training based on current national guidelines—a prospective controlled study on transferability

IntroductionMusculoskeletal ultrasound (MSUS) is integral to routine clinical diagnostics for musculoskeletal and joint disorders. This study aims to establish and validate a sonography course tailored to undergraduate medical students acquiring MSUS-specific skills at a German university.MethodsA blended learning training concept, comprising 24 instruction sessions of 45 min each, was designed based on the current national guidelines of the German Society for Ultrasound in Medicine (DEGUM). This program was integrated into the clinical phase of the undergraduate students’ medical education. The self-perceived improvement in competency and the effectiveness of the course design were evaluated using a a 7-point Likert scale questionnaire. Objective learning success was evaluated via a written test and a “Direct Observation of Practical Skills” practical exam. Control groups included medical students without MSUS training (control group 1) and doctors who had completed DEGUM-certified basic MSUS courses (control group 2). Both control groups completed the written test, while control group 2 also took the practical final exam. The study involved 146 participants: 56 were allocated to the study group, 44 to control group 1, and 46 to control group 2.ResultsThe study group rated their skills significantly higher after the course (p < 0.01). Participants expressed high satisfaction with the course design, the teaching materials, and the teachers. The study group's performance on the final written test was comparable to those of control group 2 (p = 0.06) and significantly superior to control group 1 (p < 0.001). Additionally, the study group’s performance on the practical final exam was not significantly different from control group 2 (p = 0.28), with both groups achieving scores exceeding 80%.ConclusionBoth subjective and objective measures of learning suggest that an MSUS course designed for postgraduates can be effectively adapted for undergraduate medical students. Incorporating MSUS training into the clinical curriculum is recommended to enhance future medical professionals' educational experience and practical skills.